The long range goal of the proposed work is to defme the molecular components and mechanisms mediating Vibrio cholerae colonization and virulence protein secretion to the point where there is sufficient knowledge to intelligently incorporate this information into improved cholera vaccine strategies and antimicrobial therapies designed to inhibit these events. Most of the proposal involves analysis of the molecular mechanisms by which toxin coregulated pilus (TCP) is formed and mediates intestinal colonization. Some steps in the process by which TCP and other type 4 pili are built are linked and/or related to the process of toxin and other virulence determinant secretion by type II secretion systems. Thus further understanding of the mechanisms of type 4 pilus biogenesis could lead to the characterization of potential antimicrobial targets involved in multiple virulence pathways. We will examine the aspects of pilus biogenesis in detail. These experiments are facilitated by the development during the previous grant period of an in-frame deletion mutation in each gene encoding a component of the biogenesis apparatus and by the development of antibodies directed against most of the components of the apparatus. A second aspect of pilus biogenesis and toxin secretion to be addressed will be to undertake a more complete analysis of the prepilin processing reaction mediated by type four prepilin peptidases (TFPPs). These studies will build on the identification of the TcpJ TFPP as representative of a novel class of polytopic aspartyl proteases during the previous grant period and will focus on TcpJ interactions with TcpA prepilin substrate. Finally, the proposal focuses on the characterization of the TcpF protein. During the previous grant period, TcpF was shown to be an abundant secreted colonization factor. Secretion of TcpF is unique in that it is mediated by the pilus biogenesis apparatus. At this time, TCP and the secreted TcpF protein are the only major factors known to be required for colonization by V. cholerae, with tcpA and tcpF mutants of all epidemic serogroups and biotypes showing a 5 log decrease in colonization ability. The feasibility of incorporating TcpF into defined experimental cholera subunit vaccine formulations currently being developed will also be examined in this proposal. Taken together, the results of these studies will further our ability to design rational vaccination and therapeutic intervention strategies for cholera and other bacterial infectious diseases.